New enzymes of dopamine metabolism in the zebrafish Danio rerio

The neurotransmitter dopamine is involved in the regulation of diverse functions of the nervous system, from reward processing and motor control to cell proliferation. The zebrafish dopaminergic system is an outstanding research object for research on nervous system disorders, especially for neurodevelopmental and neurochemical studies. The present work was focused on the distinct roles of the tyrosine hydroxylase genes in the development of the zebrafish nervous system, and on the previously unexplored features of COMT enzymes and genes in the zebrafish. An antibody was generated to detect zebrafish TH2, a tyrosine hydroxylase that had remained overlooked for a long time. As the new antibody recognized both TH1 and TH2 proteins, it was used together with a validated commercial anti-TH1 antibody to map the entire dopaminergic network in the zebrafish brain and to characterize the responses of TH2-expressing cells to various stressful stimuli. TH2-expressing cell groups were found in the preoptic area (group 3b), paraventricular nucleus of the hypothalamus (group 8b) and nuclei of the lateral and posterior recesses of the caudal hypothalamus (groups 9b and 10b). Cells of groups 9b and 10b were intermingled with serotonin-containing cells, but no (serotonin+TH2)-immunopositive cells were detected. Chemical and social stress induced cell activation in distinct regions of the brain, including the caudal hypothalamus, as inferred from c-fos expression, but only a small fraction of TH2 cells was activated. Dopaminergic regulation of histaminergic neuron development was assessed in pharmacological and gene knockdown experiments. Knockdown of the th1 gene by morpholino oligonucleotide injection reduced histamine and orexin neuron numbers in zebrafish larvae, whereas th2 gene knockdown increased cell numbers in these populations. These opposite effects may be due to the different spatiotemporal expression patterns of the two th genes in the zebrafish.Wnt signaling was implicated in the regulation of cell numbers. Similar patterns of expression of zebrafish comt-a and comt-b genes and a broad distribution of COMT enzymatic activity in zebrafish tissues was demonstrated. High activity and expression levels were detected in the brain, liver, and kidney. Treatment with an inhibitor of COMT did not lead to increased dopamine concentrations in zebrafish larvae, but it shifted the balance of dopamine inactivation towards the oxidation pathway catalyzed by monoamine oxidase Dopamine degradation in the zebrafish was shown to have many features in common with that in other vertebrates. Anti-TH2 antibodies were developed and characterized as tools for imaging studies in zebrafish, and possibly in other fish species. Distinct roles of TH1- and TH2-producing cells in the regulation of brain development were revealed. The thesis demonstrated that enzymes with similar biochemical functions in dopamine synthesis but different spatiotemporal expression patterns may make fundamentally different contributions to the regulation of brain development.